Growing evidence suggests that patients with Tuberous Sclerosis Complex (TSC), like other neuro-developmental disorders, have mis-wiring of neuronal connections that form during development. These defects in neuronal connectivity likely contribute to symptoms of TSC, such as cognitive deficits, autism and epilepsy. However, defective axon guidance by human neurons has only been suggested from brain imaging studies, as models to study the molecular basis for mis-guidance of developing human neurons have not been developed. To directly address these fundamental questions, we will study the development of human neurons that we differentiate from human induced pluripotent stem cells (hiPSCs) from TSC patient-derived cells and their genetically engineered counterparts. Using a series of cell behavior and molecular signaling assays, we will compare TSC2 mutant neurons with their gene-corrected, isogenic control neurons both in vitro and within 3D forebrain spheroids. We will examine changes in mTORC1 and mTORC2 signaling pathways in TSC2 mutant neurons to determine the relative contributions of each signaling pathway to neuronal development. While modulation of mTOR-dependent protein synthesis has been suggested to be required downstream of both attractive and repulsive axon guidance in several animal model systems, it is unknown if similar mechanisms function in developing human neurons. Our surprising preliminary data suggest that TSC2 functions independent of mTOR in growth cones to directly regulate the cytoskeleton to control axon guidance. In this proposal, we will determine how loss of TSC2 function alters the development of human forebrain neurons, with a current focus on axon extension and sensitivity to key axon guidance cues, two important cellular consequences of abnormal TSC2 function. We will determine the molecular mechanisms downstream of TSC2 and test functionally how these signaling pathway contribute to abnormal axon extension and guidance cue responses. Over the long term, we believe our research may help identify key druggable targets in patients with TSC.